Method for producing catalytic antibodies (variants), antigens for immunization and nucleotide sequence

ABSTRACT

A method for producing catalytic antibodies to proteins and peptides, in particular to gp120, using animals having spontaneous and induced autoimmune pathologies. The method makes it possible to create a catalytic vaccine which can when injected to a patient to exhibits adhesive properties in relation to antigen simultaneously with a destructive function, thereby suspending the progression of disease. The method for the autoimmunisation of animal lines SJL by fused proteins containing classical peptide epitope which develops pathology of an animal by protein fragments gp120 accompanied with an interest target catalytic antibody is disclosed. Also the method for immunising autoimmune animals by highly reactive chemical compositions which can perform a covalent selection of catalytic clones containing peptide fragments of potential resected portions gp120 is disclosed.

FIELD OF THE INVENTION

The present invention relates to biotechnology, immunology, geneticengineering, the microbiological and medicinal industries and comprisesa combined approach to the manufacture and expression of catalyticallyactive antibodies which are potential therapeutics intended to destroyprotein antigens, in particular gp120, which is the main surface proteinof human immunodeficiency virus.

PRIOR ART

It is known that catalytic antibodies targeted to physiologically activesubstances and natural objects useful in biomedicine may be designed asspecific representations of transition states of modeled chemicalconversions. U.S. Pat. No. 5,948,658 discloses an antibody designed bythe above approach and capable of specifically cleaving narcoticcocaine. In spite of a highly developed technology for the production ofmonoclonal antibodies, this approach cannot be effective in the case ofhigh molecular biopolymers, proteins, and peptides because it isdifficult to model corresponding transition states of the reaction.

The production of catalytic antibodies directly active against gp120 isdisclosed in WO 9703696; however, according to WO 9703696, the antibodyis obtained from patient blood serum, which impedes development of aunified medical technology for medical drug production.

SUMMARY OF THE INVENTION

The object of the present invention is to develop a method for producingcatalytic antibodies against proteins and peptides, in particular gp120,with the use of animals with spontaneous and inducible autoimmunepathologies, which method will make it possible to design a “catalyticvaccine” which, upon injection to a patient, is capable not only ofbinding the antigen but also of destroying it thus inhibiting thedevelopment of disease.

According to one embodiment, the present invention provides a method forproducing catalytic antibodies with the use of animals geneticallypredisposed to develop spontaneous and induced autoimmune pathologies.Mice are used as the animals with spontaneous and inducible autoimmunepathologies. The used mice belong to strains for which immunization withmyelin basic protein or its fragments designated in the literature as“encephalitogenic peptides” or “encephalitogenic epitopes” can inducethe development of experimental autoimmune encephalomyelitis. Theanimals are administered with a fusion protein consisting of myelinbasic protein or its fragments and a potential substrate of catalyticantibody or a fragment of the potential substrate. The potentialsubstrate is gp120 (surface glycoprotein of HIV-1) or its fragments. Thepresent invention also provides variants of chimeric proteins containingthe fragments of the gp120. These proteins are used as antigenicsubstrates to elicit the abovementioned catalytic antibody. Also saidantigenic substrates can be used as immunogens to elicitbinding/neutralizing antibodies

The present invention provides a protein comprising amino acid sequence(I): TEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLSCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGTRPVVSTQLLLNGSLAEEEVVIRSVNFTDNAKTIIVQLNTSVEINCTHCNISRAKWNNTLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQIINMWQKVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSNNESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKThe present invention also provides as a variant of the abovementionedprotein comprising amino acid sequence (I) a fusion protein having thefollowing common amino acid sequence structure (II): Z₁-X-Z₂, wherein Z₁is a sequence of from 0 to 19 amino acid residues and Z₂ are a sequenceof from 0 to 50 amino acid residues, and if Z₁ or Z₂=zero amino acidresidues, then Z₁=—H (hydrogen) and/or Z₂=—COOH (carboxygroup);X is the amino acid sequence (I).

Z₁ may be presented by a pair of amino acids, for example, by Met-Ala ora sequence of amino acids facilitating secretion of the said protein tothe extracellular space (“signal sequence”); for example, it may bebacteriophage pIII periplasmic signal 18-amino acid sequence (i.e.MKKLLFAIPLVVPFYSHS) or antibody heavy chain 19-amino acid signal peptide(i.e MNFGLRLIFLVLTLKGVQC

Z₂ may be presented by a short protein containing, for example,histidine clusters and/or the fragments of Myelin Basic Protein (MBP).

Preferred Z₂ amino acid sequences are the following:

LDPNSSSVDKLAAALEHHHHHH (this 22-amino acid sequence comprises, forexample, flexible polylinker and 6-histidine cluster);LDPNSSSVDKLAAAVVHFFKNIVTPRTPPPS (this 31-amino acid sequence comprises,for example, polylinker and a part of amino acid sequence of MBP(particularly, VVHFFKNIVTPRTPPPS); LDPHHHHHH (this 9-amino acid sequencecomprises, for example, short polylinker and histidine cluster);

GSGEQKLISEEDLNSSSVDKLAAAVVHFFKNIVTPRTPPPS (this 41-amino acid sequencecomprises, for example, short polylinker GSG, 10-amino acid segment ofimmunodominant epytope of human c-myc 62 protein EQKLISEEDL, a flexible[olylinker and and a part of amino acid sequence of MBP (particularly,VVHFFKNIVTPRTPPPS);

LDPHHHHHHGSGEQKLISEEDLNSSSVDKLAAAVVHFFKNIVTPRTPPPS (this 50-aminoacidsequence comprises, for example, two short rigid 3-aminoacid linkers,6-histidine cluster, 10-amino acid segment of immunodominant epytope ofhuman c-myc 62 protein EQKLISEEDL, a flexible polylinker and a part ofamino acid sequence of MBP (particularly, VVHFFKNIVTPRTPPPS).

As the variant of protein of common structure II the following fusionprotein is provided by the invention:MATEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLSCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSVNFTDNAKTIIVQLNTSVEINCTHCNTSRAKWNNTLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTTTLPCRIKQIINMWQKVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSNNESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKThis fusion protein is designated hereinafter as a product of Construct16.

Also the nucleotide sequence encoding the product of Construct 16 isprovided by the invention:

ctacagaaaaattgtgggtcacagtctattatggggtacctgtgtggaaggaagcaaccaccactctattttgtgcatcagatgctaaagcatatgatacagaggtacataatgtttgggccacacatgcctgtgtacccacagaccccaacccacaagaagtagtattgagctgcaacacctctgtcattacacaggcctgtccaaaggtatcctttgagccaattcccatacattattgtgccccggctggttttgcgattctaaaatgtaataataagacgttcaatggaacaggaccatgtacaaatgtcagcacagtacaatgtacacatggaattaggccagtagtatcaactcaactgctgttaaatggcagtctagcagaagaagaggtagtaattagatctgtcaatttcacggacaatgctaaaaccataatagtacagctgaacacatctgtagaaattaattgtacacattgtaacattagtagagcaaaatggaataacactttaaaacagatagctagcaaattaagagaacaatttggaaataataaaacaataatctttaagcaatcctcaggaggggacccagaaattgtaacgcacagttttaattgtggaggggaatttttctactgtaattcaacacaactgtttaatagtacttggtttaatagtacttggagtactgaagggtcaaataacactgaaggaagtgacacaatcaccctcccatgcagaataaaacaaattataaacatgtggcagaaagtaggaaaagcaatgtatgcccctcccatcagtggacaaattagatgttcatcaaatattacagggctgctattaacaagagatggtggtaatagcaacaatgagtccgagatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggc aaagtgataact

t

Also the another variant of protein of common structure II the followingfusion protein is provided by the invention:MATEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLSCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSVNFTDNAKTIIVQLNTSVEINCTHCNISRAKWNNTLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQIINMWQKVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSNNESEIFRPCCGDMRDNWRSELYKYKVVKIEPLGVAPTKAKLDPNSSSVDKLAAALE HHHHHHThis fusion protein is designated hereinafter also as the product ofConstruct 13.

Also the nucleotide sequence encoding the product of Construct 13 isprovided by the invention:ccatggctacagaaaaattgtgggtcacagtctattatggggtacctgtgtggaaggaagcaaccaccactctattttgtgcatcagatgctaaagcatatgatacagaggtacataatgtttgggccacacatgcctgtgtacccacagaccccaacccacaagaagtagtattgagctgcaacacctctgtcattacacaggcctgtccaaaggtatcctttgagccaattcccatacattattgtgccccggctggttttgcgattctaaaatgtaataataagacgttcaatggaacaggaccatgtacaaatgtcagcacagtacaatgtacacatggaattaggccagtagtatcaactcaactgctgttaaatggcagtctagcagaagaagaggtagtaattagatctgtcaatttcacggacaatgctaaaaccataatagtacagctgaacacatctgtagaaattaattgtacacattgtaacattagtagagcaaaatggaataacactttaaaacagatagctagcaaattaagagaacaatttggaaataataaaacaataatctttaagcaatcctcaggaggggacccagaaattgtaacgcacagttttaattgtggaggggaatttttctactgtaattcaacacaactgtttaatagtacttggtttaatagtacttggagtactgaagggtcaaataacactgaaggaagtgacacaatcaccctcccatgcagaataaaacaaattataaacatgtggcagaaagtaggaaaagcaatgtatgcccctcccatcagtggacaaattagatgttcatcaaatattacagggctgctattaacaagagatggtggtaatagcaacaatgagtccgagatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagctggatccgaattcgagctccgtcgacaagcttgcggccgcactcgagcaccaccaccaccaccactga

Also the invention provides another variant of fusion protein of commonstructure II having the following amino acid sequence:MATEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLSCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSVNFTDNAKTIIVQLNTSVEINCTHCNISRAKWNNTLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQIINMWQKVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSNNESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKLDPNSSSVDKLAAAVV HFFKNIVTPRTPPPSThis fusion protein is designated hereinafter also as product ofConstruct 14.

Also the nucleotide sequence encoding the product of the Construct 14 isprovided by the invention:ccatggctacagaaaaattgtgggtcacagtctattatggggtacctgtgtggaaggaagcaaccaccactctattttgtgcatcagatgctaaagcatatgatacagaggtacataatgtttgggccacacatgcctgtgtacccacagaccccaacccacaagaagtagtattgagctgcaacacctctgtcattacacaggcctgtccaaaggtatcctttgagccaattcccatacattattgtgccccggctggttttgcgattctaaaatgtaataataagacgttcaatggaacaggaccatgtacaaatgtcagcacagtacaatgtacacatggaattaggccagtagtatcaactcaactgctgttaaatggcagtctagcagaagaagaggtagtaattagatctgtcaatttcacggacaatgctaaaaccataatagtacagctgaacacatctgtagaaattaattgtacacattgtaacattagtagagcaaaatggaataacactttaaaacagatagctagcaaattaagagaacaatttggaaataataaaacaataatctttaagcaatcctcaggaggggacccagaaattgtaacgcacagttttaattgtggaggggaatttttctactgtaattcaacacaactgtttaatagtacttggtttaatagtacttggagtactgaagggtcaaataacactgaaggaagtgacacaatcaccctcccatgcagaataaaacaaattataaacatgtggcagaaagtaggaaaagcaatgtatgcccctcccatcagtggacaaattagatgttcatcaaatattacagggctgctattaacaagagatggtggtaatagcaacaatgagtccgagatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagctggatccgaattcgagctccgtcgacaagcttgcggccgcagtagtccatttcttcaagaacattgtgacacctcgaacaccacctccatcctaa ctcgag

Also the invention provides another variant of fusion protein ofstructure II which have the following amino acid sequence:MATEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLSCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSVNFTDNAKTIIVQLNTSVEINCTHCNISRAKWNNTLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQIINMWQKVGKAMYAPPISGQIRCSSNITGLLLTRDCGNSNNESEIFRPGGGDMRDNWRSELYKYKVVKTEPLGVAPTKAKLDPHHHHHHGSGEQKLISEEDLNSSSVDKLAAAVVHFFKNTVTPRTPPPSThis fusion protein is designated hereinafter also as product ofConstruct 15 or gp120 I-IIImbp protein.

Also the nucleotide sequence encoding the product of the Construct 15 isprovided by the invention:ccatggctacagaaaaattgtgggtcacagtctattatggggtacctgtgtggaaggaagcaaccaccactctattttgtgcatcagatgctaaagcatatgatacagaggtacataatgtttgggccacacatgcctgtgtacccacagaccccaacccacaagaagtagtattgagctgcaacacctctgtcattacacaggcctgtccaaaggtatcctttgagccaattcccatacattattgtgccccggctggttttgcgattctaaaatgtaataataagacgttcaatggaacaggaccatgtacaaatgtcagcacagtacaatgtacacatggaattaggccagtagtatcaactcaactgctgttaaatggcagtctagcagaagaagaggtagtaattagatctgtcaatttcacggacaatgctaaaaccataatagtacagctgaacacatctgtagaaattaattgtacacattgtaacattagtagagcaaaatggaataacactttaaaacagatagctagcaaattaagagaacaatttggaaataataaaacaataatctttaagcaatcctcaggaggggacccagaaattgtaacgcacagttttaattgtggaggggaatttttctactgtaattcaacacaactgtttaatagtacttggtttaatagtacttggagtactgaagggtcaaataacactgaaggaagtgacacaatcaccctcccatgcagaataaaacaaattataaacatgtggcagaaagtaggaaaagcaatgtatgcccctcccatcagtggacaaattagatgttcatcaaatattacagggctgctattaacaagagatggtggtaatagcaacaatgagtccgagatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagctggatccgcaccaccaccaccaccacggttccggtgaacaaaaactcatctcagaagaggatctgaattcgagctccgtcgacaagcttgcggccgcagtagtccatttcttcaagaacattgtgacacctcgaacaccacctcc atcctaactcgag

The invention also provides a method of eliciting antibodies againstgp120 glycoprotein of human immunodeficiency virus comprisingadministering of abovementioned proteins containing in their structurethe amino acid sequence (I).

According to its another embodiment, the present invention provides amethod of eliciting catalytic antibodies against gp120 glycoprotein ofhuman immunodeficiency virus by administering to mouse of SGL strain theproduct construct 15 (also designated hereinafter as protein gp120I-IIImbp or as fusion protein gp120 I-IIImbp or as gp120 I-IIImbp).

This protein (protein gp120 I-IIImbp) comprises amino acid sequence (I),immunogenic epitope of immunodominant epitope of human p62 c-myc protein(i.e amino acid sequence EQKLISEEDL) and the 89-104 peptide of themyelin basic protein (MBP) (i.e. amino acid sequence VVHFFKNIVTPRTPPPS.The SJL mouse strain is widely used as an animal model for experimentalautoimmune encephalitis (EAE) (see, for example, Liedtke W et al,Effective treatment of models of multiple sclerosis by matrixmetalloproteinase inhibitors Annals of Neurology, 1998, July; vol.,44(N1), pp. 35-46).

As a variant of said inventive method SJL mice are immunized with anantigen containing a haptene, the hapten being a conjugate of amechanism-dependent covalent protease inhibitor with a peptide, thepeptide being a gp120 or its fragment or its fragments (substrate of thecatalytic antibody).

The hapten and its isomers and racemates used in the variant of themethod of the present invention have the following structure:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A/1. Nucleotide sequence of Construct 16. NcoI-BamHI fragment ofgp120I-III is underlined by thin firm line. NcoI, BamHI restrictionsites are highlighted by bold italic type. The pET plasmid fragmentcomprising polylinker and histidine cluster is highlighted by doubleline.

FIG. 1A/2. Amino acid sequence of product of Construct 16. The fragmentgp 120 I-III is underlined: fragment I is highlighted by firm thin line;fragment II is highlighted by wavy line; fragment III is highlighted bydouble line.

FIG. 1B/1. Nucleotide sequence of Construct 13. NcoI-BamHI fragment ofgp120I-III is underlined by thin firm line. NcoI and BamHI restrictionsites are highlighted by bold italic type. The pET plasmid fragmentcomprising polylinker and histidine cluster is highlighted by doubleline.

FIG. 1B/2. Amino acid sequence of product of Construct 13. The fragmentgp 120 I-III is underlined: fragment I is highlighted by firm thin line;fragment II is highlighted by wavy line; fragment III is highlighted bydouble line. The histidine cluster is highlighted by bold type.Unhighlighted sequence LDPNNSSSVDKLAAALE is a flexible linker.

FIG. 1C/1. Nucleotide sequence of Construct 14. NcoI-BamHI fragment ofgp120I-III is underlined by thin line. NcoI, BamHI, EcoRI, NotI and XhoIrestriction sites are highlighted by bold italic type. The pET plasmidfragment comprising polylinker is highlighted by double line. TheNotI-XhoI fragment coding of MBP amino acid fragment, is highlighted bythick line and highlighted by bold type.

FIG. 1C/2. Amino acid sequence of product of Construct 14. The fragmentgp 120 I-III is underlined: fragment I is highlighted by firm thin line;fragment II is highlighted by wavy line; fragment III is highlighted bydouble line. The MBP fragment is highlighted by thick firm line and boldtype. Unhighlighted sequence LDPNNSSSVDKLAAA is a flexible polylinker.

FIG. 1D/1. Nucleotide sequence of Construct 15 (gp120 I-IIImbp protein).NcoI-BamHI fragment of gp120I-III is underlined by thin line. NcoI,BamHI, EcoRI, NotI and XhoI restriction sites are highlighted by bolditalic type. The pET plasmid fragment comprising polylinker ishighlighted by double line. The fragment coding histidine cluster ishighlighted by bold type. The sequence coding the epitope of c62-mycprotein is highlighted by thin line and italicized. The NotI-XhoIfragment coding of MBP amino acid fragment, is highlighted by thick lineand highlighted by bold type.

FIG. 1D/2. Amino acid sequence of product of Construct 15 (fusionprotein gp120 I-IIImbp protein). The fragment gp 120 I-III isunderlined: fragment I is highlighted by firm thin line; fragment II ishighlighted by wavy line; fragment III is highlighted by double line.The histidine cluster is highlighted by bold type. The epitope ofc62-myc protein is highlighted by bold italic type. The MBP fragment ishighlighted by thick firm line and bold type. Unhighlighted sequence LDPis a rigid linker. Unhighlighted sequence GSG is a flexible linker.Unhighlighted sequence NSSSVDKLAAA is a flexible linker.

FIG. 2. Diagrams of constructs obtained with the use of pET32b vectorand used for production of corresponding recombinant protein product.

FIG. 3. Diagrams of constructs obtained with the use of pET28a vectorand used for production of corresponding recombinant protein product.

FIG. 4. Electrophoregram (A) and immunoblot (B) of different stages ofisolation and purification of the protein gp120I-IIImbp (the product ofConstruct 15). 1—total cellular proteins before induction; 2—totalcellular proteins after induction; 3—the fraction of solubleintracellular proteins; 4—soluble proteins not retained by the metalchelate column; 5—soluble proteins eluted at pH 5.0; 6—soluble proteinform preparation after chromatographic purification; 7—the fraction ofinsoluble intracellular proteins; 8—insoluble proteins not retained bymetal chelate column; 9—denaturated protein form preparation afterchromatographic purification; 10—marker.

FIG. 5. Analysis of the antigen specificity of antibodies in blood serumof SJL mice (FIG. 5A) or BALB/c (FIG. 5B) immunized with the variousprepared chimeric protein products at different doses. SJL-2 andBALB/c-1 are mice immunized with the dose of 150 μg per mouse, SJL-3 andBALB/c-2 are mice immunized with the dose of 300 μg per mouse. Group ofbars I-II indicates that mice were immunized with peptide containingfragments I-II of gp 120; group II-III indicates that immunization wasdone with the peptide containing fragments II-III of gp 120; group IIIindicates results of immunization obtained with the peptide containingfragment III of gp 120; group I-III indicates results of immunizationobtained with the peptide containing fragments I-III of gp 120

FIG. 6. The principles of fluorescent and enzymic analyses ofproteolytic activity.

FIG. 7. Determination of the proteolytic activity of antibodypreparation isolated from blood serum of SJL mice immunized with thefusion protein gp120I-IIImbp. SJL-1 are control mice. SJL-2 are miceimmunized with the dose of 150 μg per mouse. SJL-3 are mice immunizedwith the dose of 300 μg per mouse. BSA-FITC and gp120-FITC were used assubstrates.

FIG. 8. Inhibition of the proteolytic activity of antibody preparationisolated from blood serum of SJL mice immunized with the fusion proteingp120I-IIImbp. SJL-1 are control mice. SJL-2 are mice immunized with thedose of 150 μg per mouse. AEBSF: aminoethanebenzenesulfonyl fluoride.CMC: phenylalanylchloromethylketone.

FIG. 9. Antispecies antibody inhibition of the proteolytic activity ofantibody preparation isolated from blood serum of SJL mice immunizedwith the fusion protein gp120I-IIImbp. SJL-1 are control mice. SJL-2 aremice immunized with the dose of 150 μg per mouse.

Anti-IgG: rabbit polyclonal antibodies against murine IgG.

FIG. 10. Enzymatic determination of the proteolytic activity of antibodypreparations isolated from blood sera of SJL mice immunized with thefusion protein gp120I-IIImbp at different doses. A: SJL-1 are controlmice; SJL-2 are mice immunized with the dose of 150 μg per mouse; SJL-3are mice immunized with the dose of 300 μg per mouse; CBA are controlCBA mice.

FIG. 11. Changes in the expression level of surface markers of T-cellsof the immune system of SJL mice immunized with the fusion proteingp120I-IIImbp at different doses, with the recombinant proteingp120I-III, and with the encephalitogenic peptide MBP₈₉₋₁₀₄. SJL-1 arenon-immunized mice. SJL-2 are mice immunized with gp120I-IIImbp at thedose of 150 μg per mouse. SJL-3: mice immunized with gp120I-IIImbp atthe dose of 300 μg per mouse. SJL-4 are mice immunized with the peptideMBP₈₉₋₁₀₄. SJL-5 are mice immunized with the recombinant proteingp120I-III at the dose of 300 μg per mouse.

FIG. 12. Enzyme immunoassay of blood sera of SJL, MRL-lpr/lpr andNZB/NZW F1 mice immunized with peptidylphosphonate. The antigen usedwas: A—biotinylated reactive peptide; B—biotinylateddiphenylvalylphosphonate; C—methyl p-nitrophenylbiotinylphenylmethylphosphonate.

FIG. 13. Electrophoregram (A) and immunoblot (B) of polyclonalantibodies isolated from immunized mice of strains SJL (4), MRL-lpr/lpr(5) and NZB/NZW F1 (6) and covalently modified with an antigen. Lanes1-3: 10 μg of BSA, 1 μg of trypsin, and 1 μg of IgG of BALB/c mice.

FIG. 14. The positive test was considered if the signal for sample ofall mice in the corresponding group was more than three times thebackground. Five animals per group were tested.

THE PREFERRED EMBODIMENTS OF THE INVENTION

The present invention is illustrated by the following Examples:

Example 1 Development of a Genetic Construct Containing a NucleotideSequence Encoding the Fusion Protein gp120I-IIImbp for its Expression ina Prokaryotic System

1) To induce autoimmune encephalomyelitis (EAE) in SJL mice, the 89-104peptide of the myelin basic protein (MBP) was chosen, the peptide havingthe following structure: VVHFFKNIVTPRTPPPS [Sakai, K., Zamvil, S. S.,Mitchell, D. J., Lim, M., Rothbard, J. B., and Steinman, L. 1988.Characterization of a major encephalitogenic T cell epitope in SJL/Jmice with synthetic oligopeptides of myelin basic protein. J.Neuroimmunol. 19:21-32, ∥ Tan, L. J., Kennedy, M. K., and Miller, S. D.1992. Regulation of the effector stages of experimental autoimmuneencephalomyelitis via neuroantigen-specific tolerance induction. II.Fine specificity of effector T cell inhibition. J. Immunol.148:2748-2755.] and is designated hereinafter also as peptide MBP₈₉₋₁₀₄.or as “MBP protein 89-104”. The DNA sequence corresponding to saidpeptide was synthesized by PCR from two overlapping oligonucleotidesadditionally containing a stop codon and restriction sites. Theresulting DNA fragment was cloned in the pET32b plasmid using NotI andXhoI restrictases. The resulting plasmid is hereinafter designated aspET32mbp. For the accurate identification of recombinant proteins at allstages of their expression, isolation and purification, pET32bCH andpET32CHmbp constructs were engineered to contain a sequence that codesfor the 10 amino acid-long fragment of immunodominant epitope of humanp62 c-myc protein [Evan G. I., Lewis G. K, Ramsay G., Bishop J. M., //Mol. Cell. Biol. 1985, V.5(12), P. 3610-3616.], namely amino acidsequence EQKLISEEDL.

2) Numerous available publications on the structure, immunogenicity andfunctional activity of the surface protein gp120 [Hansen, J. E., Lund,O., Nielsen, J. O., Brunak, S., and Hansen, J.-E., S. 1996. Predictionof the secondary structure of HIV-1 gp120. Proteins. 25: 1-11∥ Shioda,T., Oka, S., Xin, X., Liu, H., Harukuni, R., Kurotani, A., Fukushima,M., Hasan, M. K., Shiino, T., Takebe, Y., Iwamoto, A. and Nagai, Y.1997. In vivo sequence variability of human immunodeficiency virus type1 envelope gp120: association of V2 extension with slow diseaseprogression. J. Virol. 71: 4871-4881∥ Sullivan, N., Sun, Y., Sattentau,Q., Thali, M., Wu, D., Denisova, G., Gershoni, J., Robinson, J., Moore,J., and Sodroski, J. 1998. CD4-Induced Conformational Changes in theHuman Immunodeficiency Virus Type 1 gp120 Glycoprotein: Consequences forVirus Entry and Neutralization. J. Virol. 72: 4694-4703] allowedallocation of protein regions having a relatively constant sequence andmost promising with regard to immunization. For further work, a chimericpolypeptide was chosen, which consisted of three fragments of gp120(designated as I, II and III) lacking the first, second and thirdhypervariable regions. This chimeric polypeptide (as well as therespective amino acid sequence) is designated hereinafter also as“fragment gp120 I-III”. HXB2-env gene sequence was used as the initialtemplate for the synthesis of this construct [Page, K. A., Landau, N.R., and Littman, D. R. 1990. Construction and use of a humanimmunodeficiency virus vector for analysis of virus infectivity. J.Virol. 64: 5270-5276]. Fragments I, II and III were obtained by PCRusing synthetic oligonucleotides followed by assemblage of the fragmentsusing the <<splicing by overlap extension>> approach (FIG. 1). The finalPCR product I-III (designated hereinafter as “gene of gp120 I-III”) andintermediate products I-II, II-III and III were cloned into theBlueScript plasmid, with subsequent recloning into the plasmids pET32b(FIG. 2: No. 8, 9, 10 and 12), pET32mbp (FIG. 2: No. 5), pET32bCH (FIG.2: No. 6 and 11) and pET32CHmbp (FIG. 2: No. 7) using respectiverestrictases, i.e., NcoI-BamHI for I-III, NcoI-NotI for I-II,EcoRV.-BamHI for II-III, and EcoRV\DraI.-BamHI for III. The products ofthese constructs were used to test the proteolytic activity of theantibodies against gp120 that had been obtained as a result ofimmunization.

The term “catalytic antibody” means an antibody that causes accelerationof particular chemical reaction (e.g. hydrolysis of peptide bond).Catalytic antibodies are also called “abzymes”. Proteolytic antibody hasthe ability to enzymatically cleave the substrate (antigen). In thespecification the terms “catalytic” and “proteolytic” are equivalent.

For immunization of SJL mice, in order to obtain proteolytic (catalytic)antibodies against gp120 glycoprotein, the final construct based onpET28a vector was engineered (FIG. 3: No. 15). The fragment NcoI-XhoIfrom the construct 7 were recloned into pET28a at the respectiverestriction sites.

For immune response testing and antigenicity assay, additionalconstructs basing on pET28a vector (FIG. 3: No 13, No 14, No 15, No 16)including the constructs comprising the gene of gp120I-III but nosequences encoding mbp peptide and the epitope of c-myc concurrently wasobtained in a similar way as well as the protein product correspondingof these construct. Also the protein product of final construct(Construct No. 15) i.e. gp120mbp and these additional proteins weretested for ability to induce classical antibodies against gp120 inconventional laboratory animals.

A DNA “coding sequence” or a “sequence encoding” a particular protein orpeptide, is a DNA sequence which is transcribed and translated into apolypeptide in vitro or in vivo when placed under the control ofappropriate regulator elements. The boundaries of the coding sequenceare determined by a start codon at the 5′-terminus and a translationstop codon at the 3′-terminus. A coding sequence can include, but is notlimited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomicDNA sequences from eukaryotic (e. g., mammalian) DNA, and even syntheticDNA sequences. A transcription termination sequence will usually belocated 3′ to the coding sequence.

As used herein the terms “fusion protein” and “fusion peptide” are usedinterchangeably and encompass “chimeric proteins and/or chimericpeptides” and fusion “intein proteins/peptides”. A fusion proteincomprises at least fragment gp120 I-III of the present invention joinedvia a peptide bond to at least a portion of another protein. Forexample, fusion proteins can comprise a marker protein or peptide, or aprotein or peptide that aids in the isolation and/or purification and/orantigenicity and/or immunogenicity of a fragment gp120 I-III of thepresent invention

The constructs basing on pET28a allow to obtain the protein product withthe vector-encoded N-terminal amino acid sequence MA. Using differentcloning vectors, the inventors also obtained analogous series ofchimeric protein products where the initial MA dipeptide was deleted orreplaced:

by another dipeptides as KK RR, RK, ED, EN, DD, AS, AT, AA, TT, SS, NNor

by one or another prokaryotic leader peptide which widely used inrecombinant proteomics (for example, Choi J. H, Lee S Y., Secretory andextracellular production of recombinant proteins using Escherichia coli.Applied Microbiology and Biotechnology, 2004, June, vol. 64, (N5), pp625-35). including, for example, protein III of bacteriophages fd, f1,m13 (i.e. MKKLLFAIPLVVPEYSHS) or;

by one or another of eukaryotic leader peptides which widely used inrecombinant proteomics (Ladunga I. Large-scale predictions of secretoryproteins from mammalian genomic and EST sequences. Current Opinion inBiotechnology, 2000, February, vol. 11(N1), pp 13-8) including, forexample, eukaryotic leader peptide of IgG H (heavy) chain(MNFGLRLIFLVLTLKGVQC). Subsequent immunizations of animals made with thesaid proteins modified with respect to the initial construct containedthe MA dipeptide indicated that the said modifications had nosignificant influence on the ability of the chimeric protein to elicitspecific (including, correspondingly, proteolytic) humoral immuneresponse against full-length gp120 and gp120 fragment(s) includingfragment gp120 I-III or chimeric proteins including proteins comprisinggp120 fragment(s) including fragment gp120 I-III.

3) The preparation of electrocompetent cells, transformation,restrictase treatment, ligation, PCR, and DNA electrophoresis werecarried out according to standard methods [Sambrook J., Fritsch E. F.,Maniatis T. // Molecular Cloning: A Laboratory Manual. New York, ColdSpring Harbor Laboratory Press, 1989.].

Example 2 Expression, Isolation and Purification of the Fusion Proteingp120I-IIImbp

The fusion protein was expressed in T7-lysogenated E. coli cells (thestrain BL21(DE3) was used in the present example). The protein wasexpressed as follows:

1. Competent cells are transformed with 0.1 μg of plasmid according toItem 3 of Example 1 by electroporation and seeded onto a Petri dishcontaining 30 μg/ml Kanamycin and 2% glucose. Bacterial colonies aregrown for 12-14 h at 30° C. 2. The colonies are completely suspended in1 l of bacterial medium 2×YT containing 30 mg/ml Kanamycin and 0.1%glucose.

3. The cell culture is grown at 30° C. under adequate aeration up to thedensity of 0.6-1 OU but not longer than three hours; then IPTG is addedto make 1 mM and induction is carried out for 3 h at 30° C.

Fusion Protein Isolation and Purification

The fusion protein gp120I-IIImbp was isolated under denaturatingconditions as follows:

1. The cell culture is centrifuged at room temperature for 10 min at5000 rpm; the sediment is suspended in 1/50 of the initial volume in 50mM Tris-HCl, pH 8.0; lysozyme and Triton-X100 are added to make 0.1mg/ml and 0.1%, respectively; and the mixture is incubated for 30 min at30° C.

2. The lysate is cooled to 0° C., sonicated up to the disappearance ofviscosity, and centrifuged for 40 min at 20000 rpm.

3. The sediment is carefully suspended in 1/200 of the initial volume ina buffer containing 50 mM Tris-HCl (pH 8.0), 1 mM EDTA-Na, and 1%Nonidet P-40, centrifuged for 40 min at 20000 rpm, resuspended inchromatographic Buffer A and centrifuged under the same conditions.

4. The sediment is suspended in chromatographic Buffer A (50 mMNaH₂PO₄—Na₂HPO₄, 300 mM NaCl, and 6M urea, pH 8.0), incubated on ice for1 h, and centrifuged for 10 min at 20000 rpm.

5. The supernatant is applied to a metal chelate column equilibratedwith Buffer A at the rate of 10 column volumes per hour, and the columnis washed with Buffer B (50 mM NaH₂PO₄—Na₂HPO₄, 300 mM NaCl, and 6 Murea, pH 7.0) at the rate of 30 column volumes per hour up to thediscontinuation of baseline migration.

6. Elution is carried out with Buffer B (50 mM NaH₂PO₄—Na₂HPO₄, 20 mMMES-NaOH, 300 mM NaCl, and 6 M urea, pH 5.0) at the rate of 30 columnvolumes per hour, after which the column is equilibrated with Buffer Afor the second time, and immobilized metal ions and retained proteinsare removed with Buffer A containing 100 mM EDTA, pH 8.0.

7. The eluate fractions are analyzed by gel electrophoresis andcombined, and proteins are precipitated by dialysis against deionizedwater at room temperature.

8. The protein precipitate is separated by centrifugation at 4000 rpmfor 10 min, washed with 70% ethanol, suspended in a minimal volume of70% ethanol, sonicated up to the discontinuation of particlesedimentation, and stored as suspension at +4° C. in sterilepolypropylene tubes.

Fusion Protein Analysis

To confirm the identity and purity of the resulting preparations of thefusion protein gp120I-IIImbp, the following characteristics of theprotein were determined:

1. The electrophoretic purity of the protein was determined by Method 1and was found to be 97%.

2. The immunoreactivity with antibodies against c-myc epitope wasdetermined by Method 2, and the protein was found to be immunoreactive.

3. The molecular weight (Da) by mass spectrometry was determined byMethod 3 and was found to be 42307 Da, the calculated value being 42075at the tolerated error of ±2.5%.

4. The specific sorption of the protein (%) by metal chelate sorbent wasdetermined by Method 4 and was found to be >95%.

Analytical Methods:

1. Electrophoregram Densitometry.

Denaturing electrophoresis of proteins was carried out according toLaemmli using 6 M urea solution in the concentrating and fractionatinggels. Gel staining was carried out with Coomassie blue R-250 usingcontrast enhancing with a cuprum salt. Densitometry was performed with adensitometer or computer assisted plate scanner, with subsequentelectrophoregram digitalization and analysis (FIG. 4A).

1. Two-component gel is prepared to have the following composition:

-   -   Upper gel: 6.66% of acrylamide/bis-acrylamide at a 29/1 ratio,        0.1% sodium dodecyl sulphate, 0.125 M Tris-HCl, and 6 M urea, pH        6.8.    -   Lower gel: 10% of acrylamide/bis-acrylamide at a 29/1 ratio,        0.1% sodium dodecyl sulphate, 0.375 M Tris-HCl, and 6 M urea, pH        8.9.

2. Protein samples are mixed with sample buffer containing 5%2-mecaptoethanol, heated for 5 min at 100° C. and applied to gels.Electrophoresis is carried out at 25 mA until indicator dye is eluted.

3. The fractionating gel is separated and incubated for 5 min in a hotmixture of 10% ethanol and 10% acetic acid.

4. Staining is performed by gel incubation for 10 min in a hot mixtureof the following composition: 15% ethanol, 25% acetic acid, 0.3 g/lCoomassie Blue R-250, and 0.45 g/l cuprum sulphate hexahydrate.

5. After staining, the gel is subjected to multiple washings, asdescribed in Item 3, up to complete decoloration.

6. The gel is subjected to densitometry according to the densitometerspecifications. Upon electrophoregram digitization with acomputer-assisted plate scanner, the Green channel of color image orgreen light filter of the scanner is used. The electrophoregram image isanalyzed using Scion Image software by Surface Plot method. Thepreparation purity is defined as the ratio of the main peak to the sumof all the detected peaks.

2. Immunoblotting.

Immunoblotting is carried out according to the standard regimen usingblocking bovine serum albumin (BSA) solution. The hybridization bufferis supplemented with BSA (fraction V, Sigma) to make 0.5% of the finalBSA concentration (FIG. 4B).

1. Electrophoresis is carried out according to Method 2 using aprestaining marker.

2. The fractionating gel is separated, whereupon the procedure oftransference to HyBond N+ membrane (Amersham) is performed using an LKBapparatus for semidry electrotransference according to themanufacturer's specifications for 40 min at 0.8 mA/cm².

3. The membrane is blocked for 1 h with the solution of 50 mM Tris-HCl(pH 7.6), 150 mM NaCl and 5% bovine serum albumin (fraction V).

4. The membrane is washed thrice for 5 min with a deblocking solutioncontaining 50 mM Tris-HCl (pH 7.6), 150 mM NaCl and 0.05% Tween-20. Thenhybridization with the monoclonal antibody 1-9E10.2 is performed for 1 hin the solution of 50 Tris-HCl (pH 7.6), 150 mM NaCl and 0.5% bovineserum albumin.

5. Deblocking (washing) according to Item 4 is performed, and themembrane is hybridized with secondary rabbit Fc-specific anti-mouse IgGantibodies conjugated to horse radish peroxidase (Sigma Immunochemicals)under the same conditions as described in Item 4.

6. The membrane is deblocked as described in Item 4 and stained with thesolution of 50 mM Tris-HCl (pH 7.6), 3 mg/ml 1-chloro-4-naphthol and0.003% H₂O₂ for 30 min.

All the analyses are performed using primary and secondary antibodytiters of 1:10000 and 1:4000, respectively, as determined with acharacterized antigen, and a test protein is applied to electrophoresisat the dose of 0.1 μg. The presence of a possible test proteinimmunoreactivity is determined visually by the following criteria: thedevelopment of a single distinct well outlined staining zone whoseelectrophoretic mobility corresponds to that of the test protein. Whenthese criteria are met, instrumental analysis is performed.

For the final semiquantitative analysis, the densitometric evaluation ofthe intensity of the staining zone is performed by the Surface Plotmethod using Scion Image software. Test results are considered positivewhen the peak half-height is 5 times greater than the range of baselinefluctuations on the densitogram.

3. MALDI Mass-Spectrometry.

Samples are prepared for analysis as follows.

1. An aliquot of protein suspension of a minimal volume is evaporated todryness in a vacuum centrifuge.

2. The residue is dissolved in 1-5 μl of mixture of 1% aqueoustrifluoroacetic acid and 30% acetonitrile, applied to the base plateusing 2,5-dihydroxybenzoic (DHB) acid as a matrix, and analyzed.

3. A TOF MALDI mass-spectrometer, similar in performance to the VISION2000 apparatus, is precalibrated by protein reference standards (trypsinand angiotensin), and protein mass-spectra are read using internalcalibration. The masses of molecular ions are determined using VISION2000 Mass Analyzer software taking account of the performedcalibrations.

4. Specific Adsorption.

To confirm the functional properties of a protein preparation, it istested qualitatively for adsorption from solution by excess metalchelate sorbent. Tested proteins having the sequence 6×His will beimmobilized by the metal chelate sorbent at pH 8.0.

1. The required volume of the metal chelate sorbent Talon (ClontechLaboratories Inc.) is equilibrated with a buffer solution (50 mMNa₂HPO₄—NaH₂PO₄, 300 mM NaCl, and 0.1% Triton X-100) and 20 μl portionsof the 1:1 suspension are transferred to test tubes.

2. 10 μg of test protein solution is added, and the volume is adjustedto 100 μl with the buffer according to Item 1. The test tubes areincubated at shaking for 15 min and allowed to stand, after which 10 μlaliquots are taken for analysis.

3. Adsorption is considered to be complete if the measured proteinconcentration in the test sample does not exceed 0.005 μg/ml (i.e., isnot significantly different from the control when the proteinconcentration is determined by the BSA test), which corresponds to the95% absorption level.

Example 3A Immunization of Autoimmune SJL Mice with the Fusion Proteingp120I-IIImbp

SJL mice are immunized with the fusion protein gp120 I-IIImbp asfollows.

1. Five female SJL mice aged 6-8 weeks are immunized twice at a weeklyinterval with the antigen in complete Freund adjuvant having the finalM. tuberculosis concentration of 2 mg/ml and the antigen concentrationsof 1.5 mg/ml and 3 mg/ml.

2. Injections at the total volume amounting to 0.1 ml of the preparationare done subcutaneously at three sites along the back in case of thefirst immunization, and into paw pads, in case of the secondimmunization.

3. To compromise the hematoencephalic barrier, one day before the firstimmunization and two days after it the mice are additionallyintraperitoneally injected with 400 ng of pertussis toxin preparation.

4. Seventeen days after the first immunization, the mice receive aboosting peritoneal injection of the antigen in PBS at the total volumeamounting to 0.2 ml, the dose of the immunogenic protein being 50 μg permouse.

5. Concurrently with the boosting procedure, blood is taken from theorbital sinus of experimental and control (non-immunized) mice tomonitor the development of the immune response. The presence of specificantibodies in the blood serum is determined by enzyme immunoassay.

6. Twenty one days after the beginning of the experiment, the mice thathave showed the maximal antigen-specific response in immunochemicaltesting are used for splenectomy and blood sampling. The spleens areused for cell fusion in order to obtain hybridoma clones and for mRNAisolation for the subsequent cloning as phage display libraries.

Example 3B Immunization of Conventional Mice (BALB/c) with the Productsof Different Chimeric Proteins (the Protein Products of Constructs No.13-16 (see FIG. 3)

The products of Constructs Nos. 13-16 (see FIG. 3) were used as antigensfor immunization (immunogens). The control mice were treated withphisiological solution. Twenty one days after the immunization all miceare used for splenectomy and blood sampling. The BALB/c mice wereimmunized in the same manner as described in Example 3A with theexception of step 3. Control mice were treated by complete Freundadjuvant having the final M. tuberculosis concentration of 2 mg/ml andthe antigen concentrations of 1.5 mg/ml and 3 mg/ml. The all mice areused for blood sampling for estimation of immunologic response.

Example 3C Estimate of the Antigen-Specific Antibodies

To estimate the appearance of:

antigen-specific proteolytic antibodies (from SGL mice) in the course ofimmunization performed against the background of induced autoimmunepathology manifested as preclinical experimental autoimmuneencephalomyelitis,

antigen-specific antibodies (from Balb/c mice) in the course ofimmunization performed against gp120 fragments-containing proteins

the following tests are carried out:

Analysis of the Antigen Specificity and Proteolytic Activity of theObtained Polyclonal Antibody Preparations.

1. Analysis of the Antigen Specificity of Polyclonal AntibodyPreparations Obtained as from the Conventional (Balb/c) as fromAutoimmune SJL Mice.

For the initial comparative monitoring of the specific immune responseto the antigen in immunized mice, enzyme immunoassay (EIA) is used (FIG.5).

An antigen is immobilized on an immunological plate and, afterincubation with sera obtained from immunized and control mice,antigen-antibody complex is detected with Fc-specific rabbitantimouse-IgG antibodies conjugated to horse radish peroxidase. The seraof immunized and control mice are used at several dilutions (1:12 and1:48). The following recombinant proteins are used as antigens:

1) trx-gp120 I-III-CH, a fusion protein comprising E. coli thioredoxinA, a gp120 sequence lacking the first, second and third variableregions, and His₆-c-myc sequence; it is corresponding to product ofConstruct No. 6 (see FIG. 2).

2) trx-gp120 I-II-H, a fusion protein comprising E. coli thioredoxin A,the sequences of the first and second constant regions of gp120, andHis₆ sequence; it is corresponding to product of Construct No. 9 (seeFIG. 2).

3) trx-gp120 II-III-H, a fusion protein comprising E. coli thioredoxinA, the sequence of the second constant regions of gp120, with theC-terminus of the sequence starting from the third constant region, andHis₆ sequence; it is corresponding to product of Construct No. 12 (seeFIG. 2).

4) trx-gp120 III-H, a fusion protein comprising E. coli thioredoxin A,the C-terminus of the gp120 sequence starting from the third constantregion, and His₆ sequence; it is corresponding to product of ConstructNo. 10 (see FIG. 2).

5) trx-CH, a fusion protein comprising E. coli thioredoxin A and His₆sequence, which was used as the negative control to determine thenon-specific binding of obtained antibodies to these protein sequences;it is corresponding to product of Construct No. 6 (see FIG. 2).

6) Full length HIV-1 recombinant gp120 glycoprotein.

As a result of the performed experiments (FIG. 5), it has been shownthat all the antibody preparations obtained from the blood serum of miceimmunized with a fusion protein interact with the antigens. It has beenobserved as for conventional (BALB/c) mice and for autoimmune (SJLmice). The immunogenic potential of different chimeric proteins arepresented in FIG. 14. The antibody against recombinant gp120 or chimericprotein containing I, II or III fragment of gp 120 or containing itscombination (I and III), (I and II), (II and III) or (I, II, III) wasdeveloped after immunization both autoimmune and normal mice by achimeric protein having gp120 I-III amino acid chain (fragment gp 120I-III) in its structure. The additional experiments conducted with other(similar) chimeric proteins having fragment gp 120 I-III in itsstructure and optionally the short amino acid “tail” (up to 50-60 aminoacids), have shown the similar results.

2) Analysis of the Proteolytic Activity of Polyclonal AntibodyPreparations Antibody Preparations Obtained from Autoimmune SJL Mice.

To determine the proteolytic activity, the antibodies are, as apreliminary, purified by affinity chromatography using recombinantprotein G immobilized on Sepharose. The activity is detected by twodifferent methods (FIG. 6).

A. Fluorescent assay. The principle of the method, which is outlined inFIG. 6A, is based on the phenomenon of fluorescence quenching by aprotein heavily labeled with a fluorophore, which phenomenon isdescribed in literature and is mainly based on the mutual interactionsof the aromatic rings of different fluorophore molecules (e.g., becauseof intense hydrophobic and stacking contacts), and on fluorescenceenhancement by introduction of breaks into the polypeptide chain. In thepresent test, bovine serum albumin and the recombinant protein trx-gp120I-III-CH excessively labeled with fluorescein isothiocyanate (designatedhereinafter as BSA-FITC and gp120-FITC) were used as the substrates forthe proteolysis. The reaction was monitored by the fluorescenceenhancement vs. control. Trypsin devoid of contaminating chymotrypsinactivity was used as the model protease to determine the sensitivity ofthe method and to evaluate temporal signal changes depending on thesubstrate and enzyme amounts.

To determine the proteolytic activity of the tested antibodypreparations, triplicate measurements are carried out at the baselineand after incubation at 37° C. for 24 h and 48 h. The results of theseexperiments suggest the following (FIGS. 7-9):

-   First, the proteolytic activity of preparations obtained from mice    immunized with the protein gp120 I-IIImbp is increased in comparison    with control SJL mice when both substrates are used.-   Second, the increase in the antigen-specific proteolytic activity is    predominantly responsible for the total increase. With gp120-FITC,    the signal increased from ten to twenty times in comparison with    non-immunized mice, whereas with BSA-FITC, the signal increased    twofold only.-   Third, the predominant mechanism is, in this case, the    serine-dependent catalysis, because the addition of serine-reactive    irreversible inhibitors resulted in a significant reduction in the    observed rate of hydrolysis of BSA-FITC.-   Forth, IgG molecules, which are selectively removed from the    reaction by immunoprecipitation, are responsible for, at least, a    major part of the observed proteolytic activity.

Along with undisputed advantages, such as high sensitivity and thesimplicity and rapidity of measurements, this method, unfortunately, hassome drawbacks, the main of which is complex nonlinear relationshipsbetween fluorescence changes and substrate and enzyme amounts, samplevolume, buffer composition and pH, etc. resulting in difficulties in thequantitative evaluation and characterization of the enzymatic activityof polyclonal antibody preparations. Besides that, proteolytic activitydetermination by this test requires a large excess of substrate vs.enzyme, because the real amount of abzymes in the total pool ofantibodies may make a few percents or less.

With regard to the above, another method for determination of theproteolytic activity of the obtained antibody preparations was used asan alternative.

B. Enzymatic Assay.

The principle of this method of detection of proteolytic activity, whichis outlined in FIG. 6B, is based on the use of small amounts (about 1 ngper reaction) of a highly active enzyme ribonuclease A as the substrateof the proteolysis reaction. The level of ribonuclease activity, whichlinearly depends on the concentration of active RNAase A, is determinedby the acid-soluble residue method using polycytidyl acid as thepolymeric substrate of the reaction. This method of proteolytic activitydetection presumably allows achieving a significant molar excess ofenzyme vs. substrate and thus makes the conditions of the proteolysisreaction under study closer to those of the well-studied non-stationarykinetics model ([S]₀<<[E]).

To eliminate possible artefacts, all the antibody preparations understudy were tested for the intrinsic ribonuclease activity and thepresence of solution components that nonenzymatically alter the addedribonuclease activity. All the antibody preparations under study weredevoid of the intrinsic ribonuclease activity and did not alter theadded ribonuclease activity upon a short-time (10 min) incubation of thereaction.

The proteolytic activity of the tested antibody preparations wasmeasured under the following conditions: IgG concentration 0.1 mg/ml,incubation time 17 h, temperature 37° C. The preparation activitymeasured by this method was expressed as ribonuclease hydrolysis rate.

The test results presented in FIG. 10 show that proteolytic activitydecreased 1.5-2 times upon immunization of mice with gp120-I-IIImbp.These results are in a limited correspondence with the results of thefluorescent test: only the negative correlation between the proteolyticactivity and the immunogen dose used for immunization is reproduced.There is no ‘obvious’ logic indicating that the titer of catalyticantibodies or its specific catalytic activity must increase with theincrease in the antigen concentration within the described window. Lackof straightforward logic noted above is supported by the notion that thecausatives of the catalytic immune response are complex and stilllargely unknown. First, immunization with the encephalitogenic peptidecauses primarily T-cell response and tolerance breakdown onle at theappropriate genetic background. How this background influences thefurther antibody response, is unknown, however, under certainconditions, the antigens like MBP or MOG (myelin oligodendrocyteprotein) cause tolerization rather than tolerance breakdown. It istherefore difficult to expect that the following will be natural in thediscussed case 1) that the total anti-MBP autoantibody production duringartificial induction of EAE will follow general rules of “classic”immune response; 2) that the proportion of catalytic antibodies willfollow the same rules, i.e. will necessarily increase if the amount ofthe antigen increase (even though the amount of binding antibodies willindeed increase).

The discrepancy between the results of the fluorescent and enzymaticanalysis might be explained by differences between the structures of thesubstrates of proteolysis. Presumably, RNAase A globule compared withBSA globule has fewer sites available for proteases and abzymes. Since,upon immunization, the total serum concentration of IgG increasesmanifold by antigen-specific antibodies, the proportion of the initialproteolytic antibodies decreases, whereas the newly-formedantigen-specific proteolytic antibodies are, most likely, inefficientcatalyzers of proteolytic cleavage of RNAase A.

3. Monitoring of Immune Response Development and Experimental AutoimmuneEncephalomyelitis Induction.

To characterize the features of immune response development uponimmunization with a fusion protein comprising the neuroantigen MBP, acomparative analysis of specific surface markers expression inT-lymphocytes from SJL mice that were not immunized (control), from miceimmunized with the synthetic peptide MBP₈₉₋₁₀₄, recombinant fusionprotein gp120I-IIImbp (the product of Construct No. 15 (see FIG. 3) attwo different doses and recombinant protein gp120I-III (the product ofConstruct No. 13 (see FIG. 3) was carried out (FIG. 11). All theimmunizations were performed in parallel under identical conditions asdescribed above. Twenty one days after the beginning of an experiment,CD4+ T-lymphocytes isolated from two mice of each group were analyzed byflow cytometry. A specific feature of SJL mice was initially low CD8+T-cell counts. Also, changes in the expression of the following surfacemarkers important for immune response development have been studied:CD11a, CD44, CD45RB, and CD62-L. The results presented in FIG. 11suggest that in case of immunization with the peptide MBP and, also,with fusion proteins comprising this antigen, a fully developed T-cellimmune response (memory cells appeared) was induced in mice by day 21after the immunization, whereas in case of use of solely gp120I-III asthe antigen, the immune response was still developing.

The similar results were obtained in SJL mice for products of ConstructsNos 14 and 16, correspondingly).

The obtained data provide an evidence of the enhancement of immuneresponse development when the autoantigen MBP is used and ofT-lymphocyte activation typical of experimental autoimmuneencephalomyelitis development.

Thus, the above Example shows that, upon immunization of SJL mice withthe fusion protein gp120I-IIImbp, antigen-specific proteolyticantibodies are generated against the background of the preclinical stageof induced experimental autoimmune encephalomyelitis.

Example 4 Synthesis of Reactive Phosphonate Derivative of PeptideFragment of gp120

At the first stage, aminoalkylphosphonates protected at their free aminogroup are synthesized in the reaction of co-condensation oftriphenylphosphite, isobutanal, and benzylcarbamate. To this end, themixture of triphenylphosphite, isobutanal, and benzylcarbamate, 0.1 moleeach, in 15 ml of glacial acetic acid is stirred for about 1 h untilheat generation discontinues. After that, the reaction is stirred withheating to 80° C. for 1 h. After the full completion of the reaction,volatile products are removed with a rotary evaporator under reducedpressure and heating on a water bath. The oily residue is dissolved inmethanol (180 ml) and left for crystallization at −20° C. for 3 h. Aftercrystallization, the residue of diphenyl1-(N-benzyloxycarbonyl)-aminoalkylphosphonate is harvested by filtrationand recrystallized in a minimal volume of chloroform (30-40 ml) followedby the addition of four volumes of methanol.

To obtain free amynoalkylphosphonate, the protective group is removed bytreatment of diphenyl 1-(N-benzylcarbonyl)-aminoalkylphosphonate with a33% solution of hydrogen bromide in acetic acid (15 ml per 0.1 mole) for1 h at room temperature. Volatile components are removed with a rotaryevaporator at a reduced pressure and heating on a water bath.1-(N-benzyloxycarbonyl)-aminoakylphosphonate hydrobromide iscrystallized from the resulting residue by addition of anhydrous diethylether. Free phosphonate is obtained by passing gaseous dry ammoniumthrough phosphonate hydrobromide suspension in diethyl ether until theformation of a thick precipitate of ammonium bromide discontinues andthe full blooming of the suspension is observed. The resulting ammoniumbromide is removed by filtration, and diethyl ether is evaporated on awater bath under atmospheric pressure.

To obtain the hapten Leu-Ala-Glu-Glu-Glu-Val-^(P)(OPh)₂(LAEEEV-^(P)(OPh)₂), where ^(P)(OPh)₂ means the substitution of theα-carboxylic group with diphenylphosphonate, the peptideBoc-Val-Ala-(t-Bu)Glu-(t-Bu)Glu-(t-Bu)Glu protected at its N-terminalamino group and side groups is first synthesized. The peptideBoc-Val-Ala-(t-Bu)Glu-(t-Bu)Glu-(t-Bu)Glu is fused with the phosphonatederivative of valine by mixing of 2 μmoles of the protected peptide, 2μmoles of the phosphonate, and 2 μmoles of dicyclohexylcarbodiimide in300 μl of acetonitrile and incubating for 1 h. After the completion ofthe reaction, its products are separated by reverse phase HPLC on a150×3.9-mm Waters C18 NovaPak column using 0% to 80% gradient ofacetonitrile in 20 nM potassium phosphate (pH 7.0). The resultingfractions are analyzed by mass-spectrometry (MALDI-TOF). The fractionsthat contain substances with molecular ion masses of 1145 Da ([M+H]⁺),1167 Da ([M+Na]⁺) or 1183 Da ([M+K]⁺) are combined and freeze-dried. Theresidue is dissolved in 100 μl of 100% trifluoroacetic acid andincubated for 1 h at room temperature to remove protectivetert-butyloxycarbonyl and tert-butyl groups. The deblockedpeptidylphosphonate is precipitated by addition of 10 volumes ofanhydrous diethyl ether to the reaction. The precipitate is separated bycentrifuging for 10 min at 12500 rpm, and the deblocking procedure isrepeated. The residue is air-dried and stored at −20° C.

Analysis of the Peptidylphosphonate LAEEEV-^(P)(OPh)₂

I. MALDI-TOF Mass-Spectrometry.

1. A minimal amount of dry peptidylphosphonate, to which 5 μl ofacetonitrile is added, is applied to a base plate using aqueous strongacid-free 2,5-dihytroxybenzoic (DHB) acid as the matrix, and analysis iscarried out.

2. A TOF MALDI mass-spectrometer equivalent to the VISION 2000 apparatusis precalibrated with reference standards within the 500-2000 Da m/zrange, and mass spectra of test samples are obtained using internalcalibration. Molecular ion masses are determined using VISION 2000 MassAnalyzer software with the calibration taken into account. The expectedresult of the analysis is the presence of peaks corresponding to massesof 877.36, 900.34, and 915.45±1 Da.

II. Analytical Reverse Phase HPLC.

1. To a 0.2 mg sample of peptidylphosphonate, 100 μl of 20 mM potassiumphosphate buffer (pH 7.0) containing 20% acetonitrile is added.

2. The resulting sample is administered into an injector, and gradientelution is carried out using a 150×3.9 mm Waters C18 NovaPak column forreverse phase HPLC under the following conditions:

-   buffer A is 20% acetonitrile and 20 mM potassium phosphate, pH 7.0;-   buffer B is 80% acetonitrile and 20 mM potassium phosphate, pH 7.0;-   the elution rate is 1.0 ml/min at a linear 100% A to 100% B gradient    for 20 min followed by 100% B for 10 min; and-   the chromatograms are recorded at 260 nm wavelength.

3. The peaks are integrated without correction for the baseline. Theretention time of the main peak, which has the maximal area, isdetermined, and the ratio of the main peak area to the sum of all thepeak areas is calculated. The expected result: the retention time is14.75-15.25 min; the chromatographic purity is >95%.

III. Inhibition of the Esterolytic Activity of Chymotrypsin.

1. 0.5 ml of 1 μM solution of α-chymotrypsin in a buffer containing 0.1HEPES and 0.5 M NaCl, pH 7.2, is prepared. The solution is divided intonine 50-μl aliquots, and the residue is discarded.

2. Eight dilutions of the test peptidylphosphonate in acetonitrileranging from 1000 μl to 1 μl are prepared.

3. To each of the first eight aliquots of the enzyme solution 5 μl ofcorresponding peptidylphosphonate solution are added, and 5 μl ofacetonitrile are added to the ninth aliquot.

4. The samples are incubated for 1 h at 25±5° C.

5. The samples are successively transferred to a spectrophotometer cellcontaining 450 μl of deionized water, mixed, whereupon 10 μl ofp-nitrophenylacetate solution in methanol (2.5 mg/ml) are added, afterwhich the increase in the optical density at a 400-nm wavelength isrecorded. The initial rate of the substrate hydrolysis is calculated inarbitrary units.

6. The effective inhibitor concentration is calculated. To this end, theratios of the hydrolysis rates observed with samples 1-8 to thehydrolysis rate observed with sample 9 are calculated. The effectiveinhibitor concentration is determined as the lowest concentration of thetest substance, at which the ratio of the rates of substrate hydrolysisdoes not exceed 50%. The expected result: 30 μM.

Example 5 Reactive Immunization of Mice with the Phosphonate Derivativeof a Peptide Fragment of gp120

For immunization, the reactive peptide is conjugated to themacromolecular carrier C. conholepas hemocyanin (keyhole limpethemocyanin, KLH). At the first stage, the carrier is activated withexcess bis(sulfosuccinimidylyl)suberate in PBS for 1 h at 37° C. Afterthe activation, KLH unbound to bis(sulfosuccinimidylyl)suberate isremoved from the reaction by sevenfold exhaustive ultrafiltration (withthe residual volume not more than 70 μl) using a Microcon 100concentrator (Amicon YC membrane), each time adding PBS to the residueto make 500 μl and discarding the ultrafiltrate. To the resultingsolution peptidylsulfonate solution in PBS is added whereupon thesolution is incubated for 1 h at 37° C. without stirring. The unreactedsuccinimide groups are inactivated by addition of 2 μl of2-ethanolamine. The low molecular components of the reaction are removedby sevenfold exhaustive ultrafiltration (with the residual volume notmore than 70 μl) using a Microcon 100 concentrator (Amicon YC membrane),each time adding PBS to the residue to make 500 μl and discarding theultrafiltrate. The final preparation is sterilized by filtration andstored at −20° C.

Female MRL-lpr/lpr, SJL and NZB/NZW F₁ mice aged 6-8 weeks areintraperitoneally immunized with the antigen in complete Freundadjuvant, with the total volume being 0.2 ml, at the dose of 50 μg ofthe immunogenic protein per mouse.

The second immunization is done with the same volume and at the sameantigen concentration in incomplete Freund adjuvant in 17 days after thefirst immunization. Concurrently, blood is withdrawn from the orbitalsinus of three mice of each experimental group and control non-immunizedmice of the three strains to monitor the development of the immuneresponse.

Twenty one days after the beginning of the experiment, the mice thatshowed the maximal antigen-specific response in immunological tests aresacrificed for splenectomy. Polyclonal antibodies isolated from theblood serum of these mice are analyzed for antigen specificity andcatalytic activity.

A part of the peptidylphosphonate synthesized at the previous stages isused in the reaction of conjugation to N-hydroxysuccinimide ester ofbiotin. The reaction is conducted by mixing of equimolar amounts ofpeptidyl sulfonate and activated biotin in a minimal volume ofdimethylformamide and incubation for 1 h. The biotinylated preparationsare intended for analysis of the specificity of the antibodies obtainedas a result of reactive immunization of mice.

To monitor the specific immune response to the antigen in severalimmunized mice of all the three strains, enzyme immunoassay is used.

Antibodies from the blood sera of immunized and control mice areisolated with plate-preabsorbed goat antibodies against murine IgG, withsubsequent incubation with the biotinylated antigen and detection ofantigen-antibody complexes using neutravidin conjugated to horse radishperoxidase. The blood sera of immunized and control mice are used atseveral dilutions (1:12 and 1:48). The antigens employed arebiotin-labeled starting peptidylphosphonate, biotinylatedVal-phosphonate, andnitrophenylmethyl-p-biotinylphenylmethylphosphonate, for which thespecific covalent modification of the active center of abzymes wasdemonstrated earlier. The comparative analysis has shown (FIG. 12) thatthe antibodies of the experimental mice of all the three strains, on thewhole, possess a high specificity toward the modified peptide fragmentof an antigen, do not interact under the conditions of the presentexperiment with free Val-phosphonate, and exhibit the ability tocovalently bind to a more active and less specific modifying agent. Itshould be noted that, on the average, in New Zealand hybrids the amountof antigen-specific antibodies was somewhat higher in comparison withthe two other autoimmune strains, whereas the antibodies of MRL-lpr/lprmice where more effective with regard to covalent modification.

Along with an antigen, horse radish peroxidase-conjugated rabbitantibodies against the Fc fragment of murine IgG are used to determinethe total amount of murine antibodies specifically absorbed in a platewell. This allows estimation of the proportion of antigen-specificantibodies in the total pool of class G immunoglobulins.

Further studies of the type of interaction of the obtained antibodieswith a reactive peptide were carried out with pre-purified IgGpreparations using immunoblotting. After incubation with biotinylatedpeptidylphosphonate, electrophoretic fractionation under denaturing andreducing conditions, and membrane immobilization, antigen-antibodycomplexes were detected using neutravidin conjugated to horse radishperoxidase. The results of this experiment presented in FIG. 13 suggestthat both light and heavy immunoglobulin chains capable of beingcovalently modified by the peptide were present in the preparations ofpolyclonal antibodies isolated from autoimmune mice immunized with thereactive peptide Val-Ala-Glu-Glu-Glu-Val-PO(OPh)₂.

Thus, the reactive immunization under the conditions of the presentExample has produced the following results:

-   The antibodies obtained in the course of immunization bind to    immunization antigen.-   The antibodies do not react with the “minimal” phosphonate group of    immunization antigen, which means that there is no nonspecific    interaction (or nonspecific chemical reaction) between the    antibodies under study and the free phosphonate group of    Val^(P)(OPh)₂.-   The antibodies react with the active “mechanism-dependent”    phosphonate, i.e., display the ability to react with a molecule that    has no apparent structural relation to immunization antigen but has    the ability to form covalent complexes with hydrolases.-   The antibodies form covalent complexes with immunization antigen.

In combination, the above properties suggest that in the course ofimmunization with a peptidylphosphonate whose composition corresponds toLAEEEV-^(P)(OPh)₂ epitope-specific catalytic antibodies are generated.

INDUSTRIAL APPLICABILITY

The invention may be useful in medicinal industry for manufacturingdrugs and development of method for treatment HIV infection.

1. A protein comprising amino acid sequence (I):TEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLSCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSVNFTDNAKTIIVQLNTSVEINCTHCNISPAKWNNTLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQIINMWQKVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSNNESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAK


2. The protein according to claim 1 wherein said protein has thefollowing common amino acid sequence structure (II): Z₁-X-Z₂, wherein Z₁is a sequence of from 0 to 19 amino acid residues and Z₂ is a sequenceof from 0 to 50 amino acid residues, and if Z₁ or Z₂=zero amino acidresidues, then Z₁=—H (hydrogen) and/or Z₂=—COOH (carboxygroup); X is theamino acid sequence (I).
 3. A variant of the protein of claim 2, whereinsaid variant has following structure:MATEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLSCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSVNFTDNAKTIIVQLNTSVEINCTHCNISRAKWNNTLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQIINMWQKVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSNNESEIFRPGGGDMRNWRSELYKYKVVKIEPLGVAPTKAK


4. The variant of the protein of claim 2, wherein said variant hasfollowing structure: MATEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLSCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSVNFTDNAKTIIVQLNTSVEINCTHCNISRAKWNNTLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQIINMWQKVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSNNESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKLDPNSSSVDKLAAALE HHHHHH


5. The variant of the protein of claim 2, wherein said variant hasfollowing structure: MATEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLSCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSVNFTDNAKTIIVQLNTSVEINCTHCNISRAKWNNTLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQIINMWQKVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSNNESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKLDPNSSSVDKLAAAVV HFFKNIVTPRTPPPS


6. The variant of protein of claim 2, wherein said variant has followingstructure: MATEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLSCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSVNFTDNAKTIIVQLNTSVEINCTHCNISRAKWNNTLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQIINMWQKVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSNNESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKLDPHHHHHHGSGEQKLISEEDLNSSSVDKLAAAVVHFFKNIVTPRTPPPS


7. A nucleotide sequence encoding the protein of claim 3:ccatggctacagaaaaattgtgggtcacagtctattatggggtacctgtgtggaaggaagcaaccaccactctattttgtgcatcagatgctaaagcatatgatacagaggtacataatgtttgggccacacatgcctgtgtacccacagaccccaacccacaagaagtagtattgagctgcaacacctctgtcattacacaggcctgtccaaaggtatcctttgagccaattcccatacattattgtgccccggctggttttgcgattctaaaatgtaataataagacgttcaatggaacaggaccatgtacaaatgtcagcacagtacaatgtacacatggaattaggccagtagtatcaactcaactgctgttaaatggcagtctagcagaagaagaggtagtaattagatctgtcaatttcacggacaatgctaaaaccataatagtacagctgaacacatctgtagaaattaattgtacacattgtaacattagtagagcaaaatggaataacactttaaaacagatagctagcaaattaagagaacaatttggaaataataaaacaataatctttaagcaatcctcaggaggggacccagaaattgtaacgcacagttttaattgtggaggggaatttttctactgtaattcaacacaactgtttaatagtacttggtttaatagtacttggagtactgaagggtcaaataacactgaaggaagtgacacaatcaccctcccatgcagaataaaacaaattataaacatgtggcagaaagtaggaaaagcaatgtatgcccctcccatcagtggacaaattagatgttcatcaaatattacagggctgctattaacaagagatggtggtaatagcaacaatgagtccgagatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggc aaagtgataactggatcct


8. A nucleotide sequence encoding the protein of claim 4:ccatggctacagaaaaattgtgggtcacagtctattatggggtacctgtgtggaaggaagcaaccaccactctattttgtgcatcagatgctaaagcatatgatacagaggtacataatgtttgggccacacatgcctgtgtacccacagaccccaacccacaagaagtagtattgagctgcaacacctctgtcattacacaggcctgtccaaaggtatcctttgagccaattcccatacattattgtgccccggctggttttgcgattctaaaatgtaataataagacgttcaatggaacaggaccatgtacaaatgtcagcacagtacaatgtacacatggaattaggccagtagtatcaactcaactgctgttaaatggcagtctagcagaagaagaggtagtaattagatctgtcaatttcacggacaatgctaaaaccataatagtacagctgaacacatctgtagaaattaattgtacacattgtaacattagtagagcaaaatggaataacactttaaaacagatagctagcaaattaagagaacaatttggaaataataaaacaataatctttaagcaatcctcaggaggggacccagaaattgtaacgcacagttttaattgtggaggggaatttttctactgtaattcaacacaactgtttaatagtacttggtttaatagtacttggagtactgaagggtcaaataacactgaaggaagtgacacaatcaccctcccatgcagaataaaacaaattataaacatgtggcagaaagtaggaaaagcaatgtatgcccctcccatcagtggacaaattagatgttcatcaaatattacagggctgctattaacaagagatggtggtaatagcaacaatgagtccgagatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagctggatccgaattcgagctccgtcgacaagcttgcggccgcactcgagcaccaccaccaccaccactga


9. A nucleotide sequence encoding the protein of claim 5:ccatggctacagaaaaattgtgggtcacagtctattatggggtacctgtgtggaaggaagcaaccaccactctattttgtgcatcagatgctaaagcatatgatacagaggtacataatgtttgggccacacatgcctgtgtacccacagaccccaacccacaagaagtagtattgagctgcaacacctctgtcattacacaggcctgtccaaaggtatcctttgagccaattcccatacattattgtgccccggctggttttgcgattctaaaatgtaataataagacgttcaatggaacaggaccatgtacaaatgtcagcacagtacaatgtacacatggaattaggccagtagtatcaactcaactgctgttaaatggcagtctagcagaagaagaggtagtaattagatctgtcaatttcacggacaatgctaaaaccataatagtacagctgaacacatctgtagaaattaattgtacacattgtaacattagtagagcaaaatggaataacactttaaaacagatagctagcaaattaagagaacaatttggaaataataaaacaataatctttaagcaatcctcaggaggggacccagaaattgtaacgcacagttttaattgtggaggggaatttttctactgtaattcaacacaactgtttaatagtacttggtttaatagtacttggagtactgaagggtcaaataacactgaaggaagtgacacaatcaccctcccatgcagaataaaacaaattataaacatgtggcagaaagtaggaaaagcaatgtatgcccctcccatcagtggacaaattagatgttcatcaaatattacagggctgctattaacaagagatggtggtaatagcaacaatgagtccgagatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagctggatccgaattcgagctccgtcgacaagcttgcggccgcagtagtccatttcttcaagaacattgtgacacctcgaacaccacctccatcctaa ctcgag


10. A nucleotide sequence encoding the protein of claim 6:ccatggctacagaaaaattgtgggtcacagtctattatggggtacctgtgtggaaggaagcaaccaccactctattttgtgcatcagatgctaaagcatatgatacagaggtacataatgtttgggccacacatgcctgtgtacccacagaccccaacccacaagaagtagtattgagctgcaacacctctgtcattacacaggcctgtccaaaggtatcctttgagccaattcccatacattattgtgccccggctggttttgcgattctaaaatgtaataataagacgttcaatggaacaggaccatgtacaaatgtcagcacagtacaatgtacacatggaattaggccagtagtatcaactcaactgctgttaaatggcagtctagcagaagaagaggtagtaattagatctgtcaatttcacggacaatgctaaaaccataatagtacagctgaacacatctgtagaaattaattgtacacattgtaacattagtagagcaaaatggaataacactttaaaacagatagctagcaaattaagagaacaatttggaaataataaaacaataatctttaagcaatcctcaggaggggacccagaaattgtaacgcacagttttaattgtggaggggaatttttctactgtaattcaacacaactgtttaatagtacttggtttaatagtacttggagtactgaagggtcaaataacactgaaggaagtgacacaatcaccctcccatgcagaataaaacaaattataaacatgtggcagaaagtaggaaaagcaatgtatgcccctcccatcagtggacaaattagatgttcatcaaatattacagggctgctattaacaagagatggtggtaatagcaacaatgagtccgagatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagctggatccgcaccaccaccaccaccacggttccggtgaacaaaaactcatctcagaagaggatctgaattcgagctccgtcgacaagcttgcggccgcagtagtccatttcttcaagaacattgtgacacctcgaacaccacctcc atcctaactcgag


11. A method for elicing antibodies against gp120 glycoprotein of humanimmunodeficiency virus comprising administering proteins according toclaim
 1. 12. A method for elicing antibodies against gp120 glycoproteinof human immunodeficiency virus comprising administering proteinsaccording to claim
 2. 13. A method for elicing antibodies against gp120glycoprotein of human immunodeficiency virus comprising administeringproteins according to claim
 3. 14. A method for elicing antibodiesagainst gp120 glycoprotein of human immunodeficiency virus comprisingadministering proteins according to claim
 4. 15. A method for elicingantibodies against gp120 glycoprotein of human immunodeficiency viruscomprising administering proteins according to claim
 5. 16. A method forelicing antibodies against gp120 glycoprotein of human immunodeficiencyvirus comprising administering proteins according to claim
 6. 17. Amethod for elicing catalytic antibodies against gp120 glycoprotein ofhuman immunodeficiency virus comprising administering to the SGL strainmouse the protein of claim 6.